Project Summary Acute kidney injury (AKI) is recognized as a major risk for progressive chronic kidney disease (CKD). However, the mechanism by which AKI leads to fibrogenesis and ultimately to end-stage renal disease (ESRD)2 is not well defined, Due to limited knowledge of the primary signals that drive fibrogenesis, effective therapy for CKD is a major unmet medical need. Our data indicate the novel paradigm that renal denervation can prevent renal fibrosis and inflammation in three renal fibrogenesis models: 5/6 nephrectomy (5/6Nx), unilateral ureteral obstruction (UUO) and ischemic renal injury (IRI). Our data indicate that the renal nerve-derived factor, norepinephrine (NE), signaling via ?2-adrenergic receptor (?2-AR) plays a key role in initiating fibrogenesis and inflammation and its inhibition pre-or post-injury can reduce fibrosis by about 70% in these CKD models. This finding is striking, as most experimental strategies targeting a single molecule or a pathway rarely achieve a reduction of fibrosis of more than 50%. Our overall goal of this study is to delineate the mechanisms by which NE signaling via ?2-AR induces fibrosis and determine the therapeutic potential of inhibition of the ?2-AR subtype or its downstream signaling pathways in preventing renal fibrogenesis and inflammation in the IRI model. Our preliminary studies indicate that NE signaling via ?2-AR induces the expression of angiotensinogen (AGT) in renal proximal tubular cell (RPTC) via activation of cAMP-response element-binding protein (CREB). Further, simultaneous inhibition of ?2AR subtypes A and C additively protected from inflammation and fibrosis, suggesting activation of subtype specific signaling pathways, parallel to CREB-AGT axis, that may promote interstitial fibrosis and CKD. Based on these data, our central hypothesis is that NE activates ?2AR-subtypes specific-signaling pathways to induce interstitial fibrosis and their inhibition can prevent long term sequelae of IRI. Further, ?2AR activation regulates parallel pathways including, fibroblast activation and M? infiltration, activation and M? phenotypic switching, and activates Renin-Angiotensin II Signaling (RAS) signaling pathways to promote fibrogenesis. In specific aim 1, using genetic and pharmacological approaches, we will delineate the functional and mechanistic role of the three ?2-AR subtypes (A, B and C), and the effect of their inhibition on renal fibrogenesis in the IRI-model. Using transcriptomic profile, we will identify overlapping versus specific pathways between the ?2AR subtypes and identify the signaling molecules that provides added protection after combinatorial inhibition. In specific aim 2, we will dissect out the distinct role of of ?2-AR subtype(s) cell-specific role (PTC vs. M? vs fibroblasts) in cytokine secretion in fibroblast differentiation, M? behavior or M? switching and tubular injury. In specific aim 3, we will identify the ?2-AR subtype/s that activates CREB and AGT signaling and determine whether inhibition of AGT prevents inflammation and fibrosis in the IRI model. The studies have high significance as they will define ?2-AR as a primary signaling molecule that regulates several of the key pathogenic molecules and processes implicated in the renal interstitial fibrogenesis including macrophage and fibroblast activation and RAS signaling. In this regard, our studies have immediate clinical translational potential, because ?2AR inhibitors are already in clinical use in other conditions and could be adapted rapidly to prevent the progression of fibrosis in CKD and plausibly in other organs including the liver, lung and heart.